Cotransporter proteins are membrane-bound proteins that actively transport substances into cells. For example, organic substrates such as sugars, amino acids, carboxylic acids and neurotransmitters, are transported into eucaryotic cells by sodium (Na.sup.+) cotransporter proteins. Some transport proteins have been identified, for example, Na.sup.+ /glucose and Na.sup.+ /proline transporters (Peerce and Wright, Proc. Natl. Acad. Sci. USA 8:2223-2226 (1984); Wright and Peerce, J. Biol. Chem. 259:14993-14996 (1984)), and the brain Na.sup.+ Cl/GABA transporter (Radian et al., J. Biol. Chem. 261:15437-15441 (1986)), and progress has been made in locating their active sites and probing their conformational states (Peerce and Wright, supra; Wright and Peerce, supra; Peerce and Wright, J. Biol. Chem. 259:14105-14112 (1984); J. Biol. Chem. 260:6026-6031 (1985); Proc. Natl. Acad. Sci. USA 83:8092-8096 (1986); Biochem. 26:4272-4276 (1987)).
There appears to be a gene family of sodium (Na+) dependent transporters related to the mammalian intestinal Na+/glucose cotransporter, SGLT1. Na+/glucose cotransporters have been cloned and sequenced from rabbit intestine (Hediger, et al, Nature 330:379-381 (1987)) and kidney (Coady, et al, Am. J. Physiol. 259:C605-C610 (1990)), human intestine (Hediger, et al, Proc. Natl. Acad. Sci. USA 86:5748-5752, and LLC-PK.sup.1 cells (Ohta, et al, Mol. Cell. Biol. 10:6491-6499 (1990)). All of these transporters share greater than 80% identity of amino acid sequence. The only other proteins with significant homology to the mammalian SGLT1 are bacterial Na+-dependent transporters for proline (Nakao, et al, Mol. Gen. Genet. 208:70-75 (1987)) and pantothenate (Jackowski & Alix, Bacteriol. 172:3842-3848 (1990)). This suggests that the relationship between members of this multigene family is at the level of the transport mechanism, the Na+ coupling, rather than the substrate being transported.
Defects in Na.sup.+ -driven transporters may be associated with diseases. For example, a defect in the intestinal brush border Na.sup.+ /glucose cotransporter (SGLT1) is the origin of the congenital glucose-galactose malabsorption syndrome (Turk et al. Nature 350:354-356 (1991)).
It has been difficult to clone mammalian cotransport proteins in part because of the difficulties in purification of low abundance, hydrophobic membrane proteins that constitute less than 0.2% of the membrane protein. Hediger et al., Nature 330:379-381 (1987) described a new strategy for cloning rabbit intestinal Na.sup.+ /glucose cotransporter for expression without the use of antibodies or synthetic oligonucleotide probes.
Nucleosides direct a number of important physiological activities, particularly in the mammalian cardiovascular and central nervous systems. For example, adenosine is a potent vasoactive molecule in the coronary and cerebral vessels and a modulator of potassium and calcium channels in neurons and cardiac muscle (Belardinelli et al., Prog. Cardiovasc. Diseases 32:73-97 (1989)). Furthermore, nucleosides and their analogs are known to be potent cytotoxic and anti-retroviral agents. Nucleosides and their analogs, and nucleoside transport inhibitors are being used or proposed for use as broad spectrum anti-retroviral drugs, anti-cancer drugs, for treating ischemia and reperfusion-induced cell injury in the heart, and anti-arrhythmic drugs. For example, nucleoside transporter proteins may be useful to transport drugs to treat diseases such as AIDS (see, Yarchoan and Broder, New Engl. J. Med. 316:557-564 (1987)). In addition, membrane transport proteins are involved in the regulation of the uptake of nucleosides into cells. Nucleosides are required for normal growth, metabolism and function of cells. Some cells, such as bone marrow, leukocytes and brain cells, are deficient in purine biosynthesis and dependent on uptake of preformed purines.
Phloridzin, (1-[2-.beta.-D-Glucopyranosyloxy)-4,6-dihydroxyphenyl]-3]-4-hydroxyphenyl) -1-propanone, No. 7300, p. 1163, Merck Index, 11th Edition, (1989)) a phloretin-2'-.beta.-glucoside, is a natural substance occurring in all parts of the apple tree, and is known to be a very specific inhibitor of sugar transport in the mammalian intestine and kidney (see Newey et al., J. Physiol. 169:229-236 (1963) and Diedrich, Methods in Enzymology 191:755-780 (1990)). These effects are known to be due to competitive inhibition of active, i.e. sodium dependent, sugar transport. Fifty percent inhibition is achieved by phloridzin concentrations as low as 5.times.10.sup.-6 molar. Phloridzin is very specific for the Na.sup.+ /glucose cotransporter protein because the glucose moiety is recognized by the active site on the membrane transport protein. There are no other known effects of phloridzin on biological. systems.
No other specific inhibitors of sodium dependent cotransporter proteins are known.
Nucleoside cotransporter proteins have been identified (Plageman et al., Biochim. Biophys. Acta. 947:405-554 (1988)), but have not been isolated, cloned and expressed. Therefore, these proteins are available in small quantities only from mammalian cell membranes. Thus, it would be desirable to have available a method for producing practical quantities of nucleoside cotransporter protein for use alone or with drugs that can be delivered into cells by the protein and to identify inhibitors that will specifically block the activity of the cotransporter protein.